Cell-surface proteins play a major role in cell signaling, inter-cellular communication, and cell-cell, pathogen and immune recognition events. Their value as markers indicative of disease or attractive targets for therapeutic interventions has been long recognized. Miniaturized devices have emerged as useful platforms for studying a variety of intra and extra-cellular signaling processes, the read-out relying on live-cell imaging or immunocytochemistry, and, therefore, on the availability of high-specificity antibodies and/or the need for fluorescent reporters. Proteomic technologies are capable of delivering comprehensive data from whole-cell cultures, but information related to protein localization, abundance, and the presence of posttranslational modifications, protein isoforms or protein mutations is often lost. To address these limitations, the objective of this proposal is to develop the first platform that will combine the cell-handling capabilities of microfluidics with the power of mass spectrometry (MS) detection to advance a technology that can track changes in the cell-surface proteome in response to various stimuli. Aim 1 will focus on the development of a lab-on-a-chip platform that will enable cell capture, stimulation, enzymatic release of cell- surface proteins, enzymatic digestion and unambiguous MS-based identification of protein receptors and antigens, including their isoforms and possible mutated sequences. Aim 2 will focus on the optimization of the device for achieving optimal stimulation conditions of cells loaded on the chip, achieving the necessary detection limits for cell-surface receptor proteins, and developing a quantitative approach for assessing changes in the abundance of these receptors. Aim 3 will focus on demonstrating the microfluidic device for the identification of cell-surface processes that are involved in GPCR transactivation of EGFR signaling, and the initiation of cell cycle processes that support the proliferation of Her2+ cancer cells. Relevance to human health. The proposed microfluidic technology will facilitate the observation of receptor activation/transactivation and the remodeling of the cell- surface proteome to reveal new cell signal initiation mechanisms and prospects for developing treatment strategies that work in synergy with conventional drug targeting. It will also enable the identification of cell-surface antigens with biomarker potential.